Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats

Safety assessment of propyl-propane-thiosulfonate (PTSO): 90-days oral subchronic toxicity study in rats Autores: Antonio Cascajosa Lira1, Ana Isabel Prieto1, Alberto Baños2, Enrique Guillamón2, Rosario Moyano3 , Angeles Jos1*, Ana M. Cameán1. 1Area of Toxicology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain. 2 DMC Research Center SLU, Camino de Jayena s/n, Alhedin, 18620 Granada, Spain. 3 Department of Pharmacology, Toxicology and Legal and Forensic Medicine, University of Cordoba, Campus de Rabanales Carretera Madrid-Cadiz s/n *Corresponding author: Ángeles Jos Gallego Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla. C/ Profesor García González, nº 2. C.P. 41012, Seville. Spain. E-mail address: [email protected] Manuscript Title Page - A 90 days oral toxicity study was conducted with propyl-propane-thiosulfonate - PTSO did not induce toxic effects at the highest dose assayed (55 mg/Kg/day) - NOAEL for PTSO was estimated to be ≥ 55 mg/Kg/day Highlights (for review) Abstract Propyl-propane-thiosulfonate (PTSO) is one of the main organosulfur compounds present in Allium essentials oil. Different applications in the food sector have been proposed for PTSO, such as food and feed additive and as active packaging. However, the authorization of its use depends on its toxicity profile. Thus, as a part of its safety assessment, in this work a repeated dose 90-day oral toxicity study has been conducted for the first time in rats following the OECD guideline 408. PTSO was administered to groups of 10 male and 10 female rats at dose levels of 0, 14, 28, and 55 mg/Kg/day. No clinical signs or mortality and no changes in body weight, food consumption and feed conversion efficiency were detected through the study. Moreover, no treatment-related changes in hematological and biochemical parameters were observed, for either sex or dose groups. The histopathology study performed revealed no differences in organ weights, and no morphological and histopathological changes were observed. Based on these results, the no-observed-adverse-effect level (NOAEL) of PTSO was judged to be ≥ 55 mg/Kg/day for both sexes. Keywords: Subchronic toxicity; 90-day; Organosulfur compounds; Allium; Propylpropanethiosulfonate Abstract 1 1. Introduction Essentials Oils (EO) extracts from plants have been the focus of numerous studies due to their potential in the pharmaceutical and food industries. Several works and patents have been developed specifically for the application of EO and their components into the food sector (Ribeiro-Santos et al., 2017; Llana-Ruiz- Cabello et al., 2015; Maisanaba et al., 2017). Globally, their relatively safe status, properties and acceptance by consumers which demand natural compounds to replace synthetic ones have piqued the interest of industries and consumers (Sacchetti et al., 2005; Benkeblia and Lanzotti, 2007; Debiagi et al., 2014). Among those beneficial plants, Allium sp. is a genus wellknown for its antimicrobial, antiviral, antiprotozoal, antifungal or antioxidant properties (among others). These properties are mainly due to their content of organosulfur compounds (OSC), which are secondary phytochemical metabolites, biosynthesized for defensive purposes against biotic and abiotic stressors. They are mainly formed for the action of the enzyme alliin (stored in vacuoles) on cytoplasmic compounds like alk(en)yl cysteine sulfoxides (ACOs) once the vegetable tissue is hurt (Putnik et al., 2019) (Fig. 1). Otherwise, these compounds are not available in intact cells as they are toxic for the plant (Ramirez et al., 2017). One of the main components of Allium essential oil is propyl-propane-thiosulfonate (PTSO), that corresponds to the molecular formula C6H14O2S2 (Fig. 1). This compound has been stabilized and characterized by DMC Research Center SLU ( Granada, Spain) to be used for different applications, taking advantage of its beneficial properties. Thus, this product has been reported to show mainly antioxidant and antimicrobial activities, being able to inhibit the growth of Gram (–) and Gram (+) bacteria as well as molds and yeast (Peinado et al., 2012, 2013; Llana-Ruiz-Cabello et al., 2015). Its antibacterial activity in vitro in humans has also been demonstrated (Sorlozano-Puerto et al., 2018). Manuscript for revision (CLEAN VERSION) Click here to view linked References 2 Proallium AP®, a commercial Allium sp. extract with a 14.5% PTSO content, has been proposed as a biopreservative in active food packaging for human food commodities mainly due to its antioxidant and antimicrobial activity (Llana-Ruiz- Cabello et al., 2018). The packaging material used in these systems can incorporate components intended to be released into the food from the package, allowing foods to arrive at the consumers with their original or enhanced organoleptic properties, with longer shelf-life and safety (Ribeiro-Santos et al., 2017). Previously, another study carried out by Seydim & Sarikus, (2006) tested the antimicrobial activity of garlic EO in combination with oregano EO in films made with whey protein isolate, and showed antimicrobial activity in a concentration of 4% (w/v). But the incorporation of garlic EO and their components in active food packaging can result in a higher human exposure and consequently, more research is needed to stablish the safety concentration. An additional proposed application for PTSO is as sensory additive in animal nutrition, improving the palatability of feed, and also as a zootechnical additive, being an alternative to the use of antibiotics (Peinado et al., 2012), contributing to reduce resistance generated by their excessive use in livestock. Since the ban in the European Union (EU) of the use of antibiotics as growth promoters, the search for new alternative products that ensure similar production levels and food security without generating unwanted effects, including human resistances, has been fostered, being additives of natural origin a good alternative. The regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 sets the parameters of use of the additives used in animal nutrition. Recent studies have shown that some feed additives can help animals to maintain good physiological conditions and improve animal welfare. This has led to an amendment in the abovementioned regulation on 12th June 2019, establishing new functional groups of feed additives to improve 3 physiological condition of animals. In this regard, other properties demonstrating the potential use of PTSO in animal nutrition sector are the anti-methanogenic effect described during the fermentation process in rumen (Martínez-Fernández et al. 2013 ) and the immunomodulatory and anti-inflammatory properties (Vezza et al., 2019). However, the successful development of any application of this compound must be accompanied by an extensive toxicological evaluation, that guarantees its safety for the final consumers, both humans and livestock. In this regard, the toxicological profile of PTSO has been investigated by cytotoxicity assays in human cell lines (Llana-Ruiz-Cabello et al., 2015) as well as its genotoxicity and mutagenicity in vitro (Mellado-García et al., 2015) and genotoxicity in vivo (Mellado-Garcia et al., 2016b). Furthermore, its acute toxicity in vivo has been also evaluated (Llana-Ruiz-Cabello et al., 2015). Notwithstanding, safe doses of use have been not yet established for PTSO. Thus, a repeated dose 90-days oral toxicity study in rodents of PTSO would be necessary to clearly characterize its toxicity, being also a requirement in the authorization application processes of the European Food Safety Authority (EFSA, 2012; 2016a; 2016b; 2017). The aim of the present study is, therefore, to further explore the toxicity potential of PTSO and, for the first time, to conduct a subchronic dietary toxicity assay of PTSO in rats following internationally recognized test guidelines (Organisation for Economic Co-operation and Development, OECD 408, 2018). Considering these facts, in the present study several parameters have been evaluated, including body weight changes, food and water consumption, feed conversion efficiency, organ weight ratios and biochemistry and hematology parameters. In addition the histopathology of various tissues has been also studied. The results from the complete assessment of this 4 subchronic study would allow to get an estimation of a non-observed-adverse-effect- level (NOAEL) of exposure to establish safety conditions for human exposure to PTSO. 2. Materials and Methods 2.1. Test item and doses preparation PTSO was supplied by DMC RC SLU (Granada, Spain) with a 96% of purity. Commercial powder neutral gelatin from pork protein (Jesus Navarro S.A., Alicante, Spain) was employed as the vehicle for the test substance in all groups including the controls. For the 90-days study, the doses were prepared daily for each animal during the 13 weeks, and on Fridays they were also prepared for the weekend. The dose was mixed in 3 mL of liquid gelatin. The volumes of PTSO (µL) to add to the gelatin depended on the dose selected for each group, and the gelatin could solidify at 4 ºC overnight. Homogeneity of the dietary dose formulations and their stability were confirmed to be at least 5 days. 2.2. Animals conditions and husbandry services The rats were supplied by Charles River laboratories S.L. (Kings, NY, USA), 40 males and 40 females of Sprague-Dawley strain. They were approximately 7 weeks old and were stabilized for an acclimatization period of 7 days during which they were examined by a veterinary surgeon. When the first week of dosage began the rats body weight mean was 320 g ± 11.3 for males and 227 ± 11.9 for females. 5 Animals were individually housed in cages type 3H with Souralit 29/12 plus (souralit S.L., Gerona, Spain) aspen wood bedding and food completely available without restriction using standard dry pellet diet for rodents Scientific (Panlab, S.L.U., Cornella de Llobregat, Barcelona, Spain). They were kept in a room with controlled conditions of hygiene behind a barrier system, a range of temperature of 21±2ºC, with a 10-15 air changes per hour, and a relative humidity between 30-70% under 12 h light/dark cycle. Each cage contained an information card which contained study code (19-CAM-11-animal number), sex, dose, group, and individual animal identification. Community tap water (EMACSA, Cordoba Water company, Córdoba, Spain), filtered and autoclaved was available ad libitum. 2.3. Study design The maximum tolerable dose (MTD) for PTSO in rats orally exposed to PTSO (gavage) was previously set at 55 mg/kg by Llana-Ruiz-Cabello et al., (2015) following the OECD 425 (2008) test guideline (oral toxicity study: Up and Down procedure), and it was used as a reference to establish the test doses. Accordingly, this dose was selected as the highest one to be tested, and also descending doses using a 2-fold interval factor according to the guideline OECD 408 (2018) recommendations: 14, 28, 55 mg/kg/day. Rats (10/sex/group) were orally administered the selected doses and the control group received only the vehicle (pork gelatine). This study was performed at the Central Service of Experimental Animals from the University of Cordoba (SAE, Cordoba, Spain) in which all animals received human care in accordance with the guidelines for the protection of animals used for the science 6 purposes (Directive, 2010/63 EU, Decision, 2012/707/UE, and RD 53/2013). All procedures have been approved by the Ethical Animal Experimentation Committee of the University of Córdoba and by the Junta de Andalucía (project nº 20/10/2015-348). 2.4. Clinical observations Each animal was observed twice daily for morbidity and mortality and once daily for clinical signs, such as changes in skin, fur, eyes or mucous membranes; secretions; changes in gait, posture, or handling response; abnormal, clonic, or tonic movements, and stereotypes or bizarre behavior. Ophthalmic examinations were performed on all animals before initiating the study and in the control and in the highest dose group at the end of treatment. 2.5. Body weight, food and water consumption These three parameters were checked weekly in order to avoid stress. The mean body weight per group and sex were calculated weekly and prior to necropsy from individual animals’ data, as well as the food and water consumption. The total food consumed per cage was recorded and the weekly mean intake per rat was calculated. The feed conversion efficiency (FCE) ratio was determined according to Escobar et al., (2015) by the ratio of food intake (g)/ weight gained (g). 2.6. Hematology and Biochemistry Blood samples were extracted from the heart by an intracardiac injection under light isofluorane anesthesia at week 13. Then, the hematological parameters were 13 The results obtained agree with those of Mellado et al. (2016b) who evaluated the safety of Proallium AP®, an Allium-based commercial product in a 90-days feeding study with rats. PTSO is actually the major organosulfur compound present in Proallium AP® (14.5%). Similarly, neither clinical signs nor any other changes on general, biochemical, hematological or histopathological parameters were detected, and the authors derived a NOAEL higher than 400 mg/Kg/day at the conditions assayed. Both studies show a good correlation as in the present study the test item had a 7-fold higher content of PTSO than Proallium® AP (100 versus 14.5%) and the NOAEL derived for Proallium was 7-fold higher. This suggests that PTSO has an important role on the toxicity of Allium extracts. Actually, a MTD of 55 mg/kg in rats for PTSO has been established as previously indicated. And higher doses tested according to the OECD 425 guideline (2000 mg/kg and 175 mg/kg) resulted in the death of the animal and evident hepatotoxicity. On the contrary, a single dose of 55 mg/kg did not induce remarkable damage (Llana-Ruiz-Cabello et al, 2015). Mellado-García et al. (2016a) observed that in rats treated with 55 mg/kg (3 doses at 0, 24 and 45h and euthanized at 48h) an increase in the glycogen storage was noticeable in the liver and also a slight degenerative process in the chief cells of the stomach. In this regard, at the dose levels assayed, histopathological lesions were absent. This could be explained by the exposure way employed in this study, using gelatin as vehicle and with the feed. In the previous trials, oral gavage with a stomach tube after a fasting period was used following the recommendations of the corresponding OCDE guidelines. The bolus could have a more deleterious effect on the gastrointestinal system as there is a direct contact. Differences in toxicity between PTSO and Proallium AP® could be explained as the toxicity shown by components of an essential oil can be modulated by the other 14 constituents by synergistic/antagonistic phenomena (Escobar et al., 2015; Pavlidou et al., 2004). The interest of PTSO in comparison to Proallium® is based on its different chemical properties (higher hydrophilia) and in its higher efficiency on its antioxidant and antimicrobial properties. Other authors have shown the potential toxicity of aqueous extracts of different medicinal plants, including Allium sativum, in Wistar rats. Thus, Sulaiman et al. (2014) administered orally to the animals 10 mg/kg of A. sativum extract for 30 days and observed alteration in the activities of marker enzymes: AST increased in liver, kidney and heart, ALT in serum and liver, and ALP activity was reduced in serum, heart, kidney and liver. They concluded that caution was required in using unrefined extracts of these herbs in traditional settings. There are scarce in vivo toxicity data regarding other OSC. Thus, Guyonnet et al. (2000) demonstrated the effects of some of them (DAS, DADS, dipropylsulfiide (DPS) and dipropyl disulfide (DPDS)) on the activation of several mutagens in male Wistar rats exposed to 1 mmol/kg by gavage for 4 days. They explained the results based on the induction of cytochrome (CYP) and phase II enzymes activities. This effect, the alteration of CYP activity, was pointed out by other authors as well (Davenport and Wargovich, 2005). Moreover, they observed hepatotoxicity induced by DAS (bile duct obstruction, hyperproliferation and focal points of necrosis) in rats gavaged daily with 200 mg/kg for 1, 4, or 8 weeks. On the contrary, 8 weeks of exposure to lower doses (50 and 100 mg/kg) did not induced liver histophatological damage. This suggest that liver could be the target organ of OSC as both, DAS (Davenport and Wargovich, 2005) and PTSO (Llana-Ruiz-Cabello et al, 2015), have shown liver toxicity when a threshold dose is exceeded. Also, Wu et al. (2001) exposed rats orally to garlic oil (GO, 200 mg/Kg) and 3 allyl compounds, DAS (20 and 80 mg/kg), DADS (80 mg/kg), and diallyl 15 trisulfide (DATS, 70 mg/kg) 3 times a week for 6 weeks and examined the antioxidation system in rat livers and red blood cells. They found that GO, DADS and DATS significantly induced the glutathione content (GSH) in blood cells but neither GO nor any of its OSC affected the GSH-related antioxidant enzymes. Hepatic GSH was not influenced by garlic components. But DADS and DATS significantly increased the activity of GSH-reductase and GSH-transferase and decreased GSH peroxidase. In the present study the hematological parameters were not influenced by the PTSO exposure and scarce scientific data dealing with PSTO are available to compare. 5. Conclusions In conclusion, the results obtained confirm the already reported safety profile of PTSO for some food applications at the conditions considered. Thus, PTSO did not promote toxic effects as seen from body weight changes, food and water consumption, feed conversion efficiency, biochemical and blood parameters as well as organ toxicity and histological examinations of main organs that could eventually be affected by its subchronic administration (90 days). NOAEL was estimated to be ≥ 55 mg/Kg/day. Conflict of Interest The authors declare that there are no conflicts of interest. 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The immunomodulatory properties of propyl-propane thiosulfonate contribute to its intestinal anti-inflammatory effect in experimental colitis. Mol. Nutr. Food Res. 63(5):e1800653. doi: 10.1002/mnfr.201800653. Wu, C.C., Sheen, L.Y., Chen, H.W., Tsai, S.J., Lii, C.K. (2001). Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells. Food. Chem. Toxicol. 39, 633-569. doi.org/10.1016/S0278- 6915(00)00171-X RBC: Erythrocyte count; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; PLT: platelet (thrombocyte) count; RDW: red blood cell distribution width; T PRO: prothrombin time; T CEF: cephalin time. F: Statistics ANOVA test; K.W: Kruskal-WallisStatistic; N.S.: Not Significant; &Significantly different in comparison to group 4 when p<0.05; # significantly different in comparison to group 3 when p<0.05. Table 3 WBC: total leukocyte count; NE: neutrophil; LY: lymphocyte; MO: monocyte; EO: eosinophil; BA: basophil; F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. DIFFERENTIAL WHITE BLOOD CELLS COUNT DATA SUMMARY MALE FEMALE Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 WBC MEAN 11.43 11.84 12.03 11.70 7.19 6.02 6.83 4.54 103/µL ST. DEV. 3.38 1.70 1.88 3.80 3.67 1.93 2.16 2.70 KW= 0.60 p=0.90; N.S. F(34.3)=1.86 p=0.16; N.S. NE MEAN 16.43 14.59 16.07 16.95 30.24 31.03 24.66 24.99 % ST. DEV. 5.07 2.80 3.53 5.88 27.30 15.46 15.28 11.31 KW=1.47 p=0.69; N.S. F(34.3)=0.35 p=0.79; N.S. LY MEAN 71.87 80.02 78.09 74.52 64.73 62.00 68.32 67.00 % ST. DEV. 14.48 4.25 5.78 8.01 26.04 17.24 14.42 12.00 KW= 5.07 p=0.17; N.S. F(34.3)=0.25 p=0.86; N.S. MO MEAN 3.01 1.52 2.87 2.94 1.44 1.07 0.75 1.57 % ST. DEV. 2.24 1.81 2.33 2.16 1.66 0.74 0.77 1.78 KW=2.71 p=0.44; N.S. KW=2.22 p=0.53; N.S. EO MEAN 3.38 3.22 2.03 4.58 3.17 6.33 4.52 6.94 % ST. DEV. 2.33 1.69 0.58 2.88 2.52 4.53 2.90 5.23 KW=6.70 p=0.08; N.S. KW=3.14 p=0.37; N.S. BA MEAN 0.57 0.70 0.95 1.07 0.41 0.54 0.80 0.51 % ST. DEV. 0.50 0.44 1.39 1.09 0.41 0.41 0.65 0.39 KW=1.42p=0.70; N.S. F(34.3)= 1.01 p=0.40; N.S. Table 3 Table 4 CLINICAL BIOCHEMISTRY DATA SUMMARY MALE FEMALE Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 GLUC MEAN 143.50 146.50 128.40 123.60 125.60 121.60 105.20 104.00 mg/dL ST. DEV. 22.73 27.44 26.31 20.30 16.64 17.20 27.59 44.93 F(36.3)=2.12 p=0.12; N.S. KW=4.90 p=0.18; N.S. UREA MEAN 35.22 31.67 33.99 35.37 30.26 31.87 34.90 34.22 mg/dl ST. DEV. 4.83 2.95 4.37 3.16 4.63 3.36 7.02 4.14 F(36.3)=1.92 p=0.14; N.S. F(36.3)=1.85 p=0.16; N.S. CREAT MEAN 0.30 0.28 0.29 0.29 0.34 0.35 0.35 0.34 mg/dL ST. DEV. 0.04 0.02 0.03 0.03 0.06 0.03 0.07 0.02 F(36.3)=0.48 p=0.70; N.S. F(36.3)=0.26 p=0.86; N.S. BILI-T MEAN 0.21 0.31 0.21 0.22 0.26 0.28 0.25 0.26 mg/dL ST. DEV. 0.02 0.32 0.02 0.03 0.03 0.02 0.03 0.01 KW=1.41 p=0.70; N.S. F(36.3)=2.15 p=0.11; N.S. CHOL MEAN 107.50 95.40 93.50 88.50** 104.20 101.80 112.40 99.20 mg/dL ST. DEV. 13.83 15.36 10.60 8.50 11.12 20.75 20.58 15.36 F(36.3)=4.25 p<0.01** F(36.3)=1.08 p=0.36; N.S. TRIGL MEAN 156.40 163.40* 131.80 123.00 78.60 104.80 84.40 77.80 mg/dL ST. DEV. 33.81 36.16 27.59 20.96 18.87 28.06 21.53 16.94 KW=10.48 p<0.05* F(36.3)=1.85 p=0.16; N.S. AST MEAN 147.52 145.76 135.39 183.73 264.52 246.91 251.94 264.62 U/L ST. DEV. 17.74 26.66 17.66 101.03 94.90 92.11 73.01 122.10 KW=3.44 p=0.33; N.S. KW= 0.39 p=0.95; N.S. ALT MEAN 30.46 38.36 33.42 31.44 38.31 35.40 37.03 35.22 U/L ST. DEV. 6.26 16.86 6.09 2.42 10.34 17.81 5.26 10.55 KW=3.12 p=0.38; N.S. KW=2.41 p=0.49; N.S. ALKP MEAN 91.20 98.20 88.30 89.60 62.00 62.90 66.40 60.60 U/L ST. DEV. 9.62 18.50 26.97 15.21 12.14 9.99 17.22 9.13 Table 4 GLUC: glucose; CREAT: creatinine; Bili-T: Bilirubin, total; CHOL:cholesterol, total; TRIGL: triglycerides; AST:aspartate aminotransferase; ALT: alanine aminotransferase; ALKP: alkaline phosphatase; ALB:albumin; TOT PROT: protein, total; Na+:sodium; K+:potassium; Ca++: calcium. F: Statistics ANOVA test; K.W:Kruskal-WallisStatistic; N.S.: Not Significant *Significantly different from control. *when p<0.01 **Significantly different from control. **when p<0.01 KW=0.56 p=0.65; N.S. F(36.3)=0.39 p=0.76; N.S. ALB MEAN 3.93 3.88 3.98 4.00 4.80 4.59 4.66 4.72 g/dl ST. DEV. 0.47 0.25 0.32 0.29 0.35 0.34 0.59 0.34 KW=1.54 p=0.67; N.S. F(36.3)=0.47 p=0.70; N.S. TOT PROT MEAN 6.24 6.26 6.26 6.20 6.46 6.78 6.78 6.54 g/dl ST. DEV. 0.34 0.48 0.37 0.29 0.44 0.35 0.68 0.51 F(36.3)=0.06 p=0.98; N.S. F(36.3)=1.05 p=0.38; N.S. Na+ MEAN 134.80 133.70 133.60 132.80 142.60 139.60 140.20 139.40 mmol/L ST. DEV. 10.40 3.59 3.24 4.08 4.62 7.12 11.64 7.78 KW=2.00 p=0.57; N.S. KW=1.65 p=0.65; N.S. K+ MEAN 7.87 8.89 7.90 8.38 9.68 12.81 14.93 10.34 mmol/L ST. DEV. 1.58 1.62 0.77 1.29 6.97 6.18 7.61 5.67 F(36.3)=1.26 p=0.30; N.S. KW=6.35 p=0.10; N.S. Ca++ MEAN 10.87 10.28 10.82 10.51 11.30 11.42 10.76 11.25 mg/dL ST. DEV. 1.04 0.66 0.64 0.47 1.06 1.26 0.87 1.36 KW=4.43 p=0.22; N.S. KW=2.34 p=0.51; N.S. Table 5 F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. * Significantly different from group 3 in comparison to group 1 when p<0.05. ORGAN WEIGHT DATA SUMMARY MALE FEMALE Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 BODY W. MEAN 633.80 623.10 611.5 625.90 BODY W. MEAN 326.80 338.00 321.10 321.30 (g) ST. DEV. 38.25 28.71 26.50 48.14 (g) ST. DEV. 11.50 29.08 23.81 18.31 F(36.3)=0.19 p=0.90; N.S. F(36.3)=1.34 p=0.29; N.S. BRAIN MEAN 2.09 2.13 2.12 2.07 BRAIN MEAN 1.96 2.03 2.01 2.06 (g) ST. DEV. 0.21 0.14 0.17 0.23 (g) ST. DEV. 0.21 0.10 0.12 0.15 KW=0.09 p=0.99; N.S. KW=2.35 p=0.50; N.S. LIVER MEAN 21.06 18.49 18.40 18.41 LIVER MEAN 8.41 9.54 8.90 8.48 (g) ST. DEV. 4.86 1.45 1.77 2.40 (g) ST. DEV. 1.36 1.09 1.08 0.83 F(36.3)=1.99 p=0.13; N.S. F(36.3)=2.21 p=0.10; N.S. HEART MEAN 2.14 1.98 1.82* 1.99 HEART MEAN 1.21 1.28 1.20 1.22 (g) ST. DEV. 0.18 0.19 0.19 0.32 (g) ST. DEV. 0.13 0.13 0.14 0.16 F(36.3)=3.11 *p<0.05 F(36.3)=0.67 p=0.59; N.S. SPLEEN MEAN 1.17 1.16 1.16 1.45 SPLEEN MEAN 0.80 0.86 0.74 0.75 (g) ST. DEV. 0.10 0.15 0.14 0.93 (g) ST. DEV. 0.11 0.19 0.07 0.09 KW=0.2426 p=0.9704; N.S. F(36.3)=1.71 p=0.18; N.S. KIDNEYS MEAN 4.05 4.10 3.99 4.06 KIDNEYS MEAN 2.13 2.30 2.15 2.06 (g) ST. DEV. 0.30 0.41 0.37 0.28 (g) ST. DEV. 0.23 0.23 0.20 0.14 F(36.3)=0.17 p=0.92; N.S. F(36.3)=2.48 p=0.08; N.S. THYMUS MEAN 0.88 0.80 0.83 0.71 THYMUS MEAN 0.67 0.70 0.66 0.60 (g) ST. DEV. 0.25 0.12 0.15 0.27 (g) ST. DEV. 0.08 0.13 0.20 0.10 F(36.3)=1.20 p=0.32; N.S. F(36.3)=1.02 p=0.40; N.S. TESTES MEAN 3.92 3.95 3.77 3.83 UTE./CERV. MEAN 0.87 0.76 1.03 0.94 (g) ST. DEV. 0.44 0.29 0.32 0.28 (g) ST. DEV. 0.24 0.17 0.81 0.32 F(36.3)=0.62 p=0.61; N.S. KW=1.32 p=0.73; N.S. EPIDIDIMS MEAN 1.88 2.05 2.36 2.07 OVARIES MEAN 0.26 0.31 0.23 0.25 (g) ST. DEV. 0.24 0.52 0.98 0.28 (g) ST. DEV. 0.0.8 0.10 0.06 0.07 KW=4.00 p=0.26; N.S. F(36.3)= 1.90 p=0.15; N.S. ADRENALS MEAN 0.11 0.13 0.12 0.12 ADRENALS MEAN 0.15 0.12 0.10 0.12 (g) ST. DEV. 0.04 0.05 0.07 0.05 (g) ST. DEV. 0.08 0.04 0.02 0.08 KW=0.72 p=0.87; N.S. KW=4.95 p=0.18; N.S. Table 5 Table 6 F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. ORGAN WEIGHT/BODY WEIGHT RATIO DATA SUMMARY MALE FEMALE Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 BRAIN MEAN 0.33 0.34 0.36 0.33 BRAIN MEAN 0.60 0.60 0.63 0.64 (%) ST. DEV. 0.03 0.03 0.06 0.04 (%) ST. DEV. 0.07 0.05 0.6 0.05 KW=4.99 p=0.17; N.S. F(36.3)=1.17 p=0.34; N.S. LIVER MEAN 3.31 2.97 3.22 2.93 LIVER MEAN 2.58 2.82 0.77 2.64 (%) ST. DEV. 0.71 0.15 0.71 0.21 (%) ST. DEV. 0.42 0.23 0.27 0.24 KW=2.03 p=0.57; N.S. F(36.3)=1.47 p=0.24; N.S. HEART MEAN 0.33 0.31 0.32 0.32 HEART MEAN 0.37 0.38 0.38 0.38 (%) ST. DEV. 0.04 0.02 0.05 0.05 (%) ST. DEV. 0.04 0.05 0.06 0.05 F(36.3)=0.64 p=0.59; N.S. KW=1.12 p=0.77; N.S. SPLEEN MEAN 0.18 0.19 0.20 0.23 SPLEEN MEAN 0.25 0.25 0.23 0.23 (%) ST. DEV. 0.02 0.02 0.03 0.14 (%) ST. DEV. 0.03 0.05 0.02 0.03 KW=2.15 p=0.54; N.S. F(36.3)=0.76 p=0.52; N.S. KIDNEYS MEAN 0.63 0.65 0.70 0.65 KIDNEYS MEAN 0.65 0.68 0.67 0.64 (%) ST. DEV. 0.05 0.05 0.15 0.06 (%) ST. DEV. 0.07 0.06 0.06 0.06 KW=1.42 p=0.70; N.S. KW=2.53 p=0.47; N.S. THYMUS MEAN 0.14 0.13 0.15 0.11 THYMUS MEAN 0.21 0.21 0.21 0.19 (%) ST. DEV. 0.02 0.02 0.05 0.04 (%) ST. DEV. 0.03 0.04 0.06 0.03 KW=4.80 p=0.19; N.S. F(36.3)=0.68 p=0.57; N.S. TESTES MEAN 0.62 0.63 0.66 0.61 UTE./CERV. MEAN 0.27 0.23 0.32 0.29 (%) ST. DEV. 0.07 0.05 0.14 0.07 (%) ST. DEV. 0.07 0.05 0.25 0.10 KW=1.16 p=0.76; N.S. KW=2.47 p=0.48; N.S. EPIDIDIMS MEAN 0.30 0.32 0.41 0.33 OVARIES MEAN 0.08 0.09 0.07 0.08 (%) ST. DEV. 0.04 0.08 0.18 0.05 (%) ST. DEV. 0.02 0.03 0.02 0.02 KW=5.544 p=0.1361; N.S. KW=3.53 p=0.32; N.S. ADRENALS MEAN 0.02 0.02 0.02 0.02 ADRENALS MEAN 0.05 0.04 0.03 0.04 (%) ST. DEV. 0.01 0.01 0.01 0.01 (%) ST. DEV. 0.02 0.01 0.01 0.02 KW=0.45 p=0.93; N.S. KW=4.49 p=0.21; N.S. Table 6 Table 7 F: Statistics ANOVA test; K.W: Kruskal-Wallis Statistic; N.S.: Not Significant. ORGAN WEIGHT/BRAIN WEIGHT RATIO DATA SUMMARY MALE FEMALE Group 1 Group 2 Group 3 Group 4 Group 1 Group 2 Group 3 Group 4 (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) (0 mg/Kg/day) (14 mg/Kg/day) (28 mg/Kg/day) (55 mg/Kg/day) N=10 N=10 N=10 N=10 N=10 N=10 N=10 N=10 LIVER MEAN 1011.66 874.10 876.04 892.82 LIVER MEAN 437.12 470.10 444.59 411.74 (%) ST. DEV. 229.58 103.29 131.13 92.16 (%) ST. DEV. 104.65 48.18 56.83 41.47 KW=4.08 p=0.25; N.S. F(36.3)=1.27 p=0.30; N.S. HEART MEAN 103.79 93.72 86.79 97.94 HEART MEAN 62.93 63.41 60.00 59.58 (%) ST. DEV. 16.99 12.97 10.89 23.79 (%) ST. DEV. 13.12 8.56 8.83 9.81 KW=6.19 p=0.10; N.S. F(36.3)=0.37 p=0.78; N.S. SPLEEN MEAN 56.36 54.78 55.10 55.42 SPLEEN MEAN 41.83 42.21 37.10 36.61 (%) ST. DEV. 7.85 6.83 7.14 7.75 (%) ST. DEV. 9.96 9.33 3.40 6.17 F(36.3)=0.084 p=0.97; N.S. F(36.3)=1.52 p=0.23; N.S. KIDNEYS MEAN 195.30 193.90 190.21 198.22 KIDNEYS MEAN 110.18 113.37 107.39 100.25 (%) ST. DEV. 22.29 27.64 30.06 26.91 (%) ST. DEV. 18.71 12.13 13.40 12.68 F(36.3)=0.15 p=0.93; N.S. KW=5.94 p=0.11; N.S. THYMUS MEAN 42.63 37.87 39.25 34.33 THYMUS MEAN 34.57 34.51 33.29 28.27 (%) ST. DEV. 13.08 6.61 7.85 12.04 (%) ST. DEV. 5.25 5.96 12.14 4.26 F(36.3)=1.13 p=0.35; N.S. KW=5.75 p=0.12; N.S. TESTES MEAN 189.99 187.00 179.45 187.33 UTE./CERV. MEAN 44.81 37.40 51.51 46.40 (%) ST. DEV. 33.59 23.50 24.34 28.69 (%) ST. DEV. 12.42 8.40 40.13 18.00 F(36.3)=0.26 p=0.85; N.S. KW=2.62 p=0.45; N.S. EPIDIDIMS MEAN 89.92 96.89 112.09 100.46 OVARIES MEAN 13.04 15.32 11.31 12.10 (%) ST. DEV. 7.49 24.61 46.97 12.46 (%) ST. DEV. 3.734 5.41 3.20 3.43 KW=3.96 p=0.27; N.S. F(36.3)=1.85 p=0.16; N.S. ADRENALS MEAN 5.42 5.40 5.91 5.75 ADRENALS MEAN 8.00 6.10 4.82 5.98 (%) ST. DEV. 2.59 2.83 3.99 2.77 (%) ST. DEV. 4.87 2.02 1.33 3.97 KW=0.50 p=0.92; N.S. KW=4.27 p=0.23; N.S. Table 7 Figure 1 Click here to access/download;Figure;figura 1. ajo.tif Figure 2 Click here to access/download;Figure;Figure 2. Body weight.tif Figure 3 Click here to access/download;Figure;Figure 3. Mean body weight gain.tif